Hydrophobic handoff for direct delivery of peroxisome tail-anchored proteins

• Published in: Nature communications Vol. 5; no. 1; p. 5790
• Main Authors: Chen, Yinxiao, Pieuchot, Laurent, Loh, Rachel Ann, Yang, Jing, Kari, Teuku Mahfuzh Aufar, Wong, Jie Yun, Jedd, Gregory
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 17.12.2014; Nature Publishing Group
• Subjects: 631/45; 631/80; Amino Acid Sequence; Animals; Epithelial Cells - cytology; Epithelial Cells - metabolism; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - metabolism; Gene Expression; Genes, Reporter; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Humanities and Social Sciences; Humans; Hydrophobic and Hydrophilic Interactions; Kidney; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Molecular Sequence Data; multidisciplinary; Neurospora crassa - genetics; Neurospora crassa - metabolism; Peroxisomes - chemistry; Peroxisomes - metabolism; Protein Binding; Protein Interaction Domains and Motifs; Protein Transport; Rats; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Red Fluorescent Protein; Science; Science (multidisciplinary); Sequence Alignment; Sequence Homology, Amino Acid
• DOI: 10.1038/ncomms6790
• ISSN: 2041-1723

Tail-anchored (TA) proteins are inserted into membranes post-translationally through a C-terminal transmembrane domain (TMD). The PEX19 protein binds peroxisome TA proteins in the cytoplasm and delivers them to the membrane through the PEX3 receptor protein. An amphipathic segment in PEX19 promotes docking on PEX3. However, how this leads to substrate insertion is unknown. Here we reconstitute peroxisome TA protein biogenesis into two sequential steps of substrate TMD engagement and membrane insertion. We identify a series of previously uncharacterized amphipathic segments in PEX19 and identify one whose hydrophobicity is required for membrane insertion, but not TMD chaperone activity or PEX3 binding. A membrane-proximal hydrophobic surface of PEX3 promotes an unconventional form of membrane intercalation, and is also required for TMD insertion. Together, these data support a mechanism in which hydrophobic moieties in the TMD chaperone and its membrane-associated receptor act in a concerted manner to prompt TMD release and membrane insertion. Delivery and insertion of tail-anchored proteins into peroxisomal membranes requires the chaperone PEX19 and its receptor PEX3. Chen et al. reconstitute this process in a cell-free reaction and show that distinct hydrophobic moieties in PEX19 are required at three separable import steps.